This grant application is a response to Topic 15-OD (ORDR)-101. Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is an inherited cardiac disease characterized by severe ventricular arrhythmias, as well as progressive replacement of healthy myocardium with fibrous and fatty tissue leading in many cases to severe heart failure and, in the absence of a heart transplant, death. ARVC has been associated with mutations in desmosomal proteins, most prominently in the gene coding for plakophilin-2 (PKP2). Much attention has been given to the changes that occur in cardiac myocytes following disruption of the desmosome. Yet, myocyte-based experimental models have failed to reproduce the fibrofatty infiltration characteristic of the disease. Here, we shift away from cardiomyocyte-centered hypotheses to propose a new paradigm: that the fibrofatty infiltration has its origins in the behavior of non-myocyte cells. Our focus centers on two stem cell populations capable of generating a cardiac fibroblast lineage: the epithelial cells that comprise the epicardium, and the hematopoietic stem cells. Through the use of novel mouse molecular genetic strategies, and cell biological assays, we will test the hypothesis that loss of desmosomal integrity in these stem cells leads to an increase in the population of fibroblasts and adipocytes in the heart, at the expense of myocardial mass. Specific aims are: 1. To assess the role of PKP2 on migration, proliferation and differentiation of epicardium-derived cells. 2. To generate a murine model of epicardium-specific conditional expression of an ARVC-relevant mutation. 3. To generate and characterize an animal model of hematopoietic stem cell-specific PKP2 deficiency. These studies offer new hypotheses and new experimental models to better understand fibroblast biology in the diseased heart. Moreover, our experiments open the tantalizing possibility that in the future, these stem-cell populations (hematopoietic;epicardial) can be targeted for treatment, to decrease the rate of loss of myocardial mass and prevent the occurrence of heart failure in patients with ARVC. Moreover, although ARVC is, indeed, a "rare disease," cardiac fibrosis is a common occurrence of major medical importance, and control of fibrosis via manipulation of the fibroblast progenitor cells remains largely unexplored. Experimental models generated under the present grant can be instrumental to reduce that particular knowledge gap. Animal models where the stem cell progeny is traceable will become available to the scientific community as a whole. These animals will be utilized on additional experiments where acquired diseases (e.g., LAD occlusion;pacing-induced hypertrophy) are modeled, thus allowing identification of the origin of the resulting fibroblasts. As such, this project is directly relevant to the small community of ARVC-afflicted patients. Yet, as it is often the case with the study of rare diseases, lessons learned from the present project are likely to find applications relevant to the much broader community of patients afflicted with heart disease, the number one killer in the United States. PUBLIC HEALTH RELEVANCE: Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is a devastating, incurable disease of the heart that can run in families. Patients suffer very severe arrhythmias and are at great risk of sudden death at a young age. As the disease progresses, the heart muscle weakens and becomes replaced with fatty and fibroid tissue, to the point where the heart is unable to pump blood. In the terminal stages (often in young adults), heart transplant becomes the only alternative. Here, we propose that part of the origins of the disease may involve a particular type of stem cells, which may produce excess fatty and fibroid cells. To understand the role of these stem cells in ARVC, and potentially manipulate their function to preserve the heart wall, are the main goals of this grant.